Bacterial biofilms are highly complex communities, composed of highly structured extracellular polymers and subpopulations of differentiated cells, such as persisters. These contribute to the resistance of bacterial biofilms to antibiotics, creating a significant issue in the treatment of infections and resulting in elevated levels of mortality and morbidity. Here, we use a microfluidic system coupled with time-lapse microscopy and fluorescent dyes for exopolysaccharide (EPS) and extracellular DNA (eDNA) to investigate how the architecture of the growing biofilm is ordered. We show that in Pseudomonas aeruginosa EPS is deposited first in the initial stages of microcolony development, but that eDNA then acts as a leading edge for further microcolony expansion. We explore how this assembly is perturbed by the introduction of different antimicrobial agents. Further, working with partners in the European Association of National Metrology Institutes (EURAMET) we are developing cross platform methods for the label-free localisation of antimicrobial agents and bacterial components within the biofilm. These platforms include 3D OrbiSIMS (secondary ion mass spectrometry) and Raman spectroscopy. Here we demonstrate the localisation of key biofilm components such as Pseudomonas Quinolone Signal (PQS) molecules in a 3D chemical map of the biofilm.


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